Aequorin is a photoprotein isolated from luminescent jellyfish Aequoria victoria.
Apoaequorin is a protoprotein which, upon binding to coelenterazine, can emit photons in the presence Ca2+. The Aequorin complex comprises a 22,514 MW Apoaequorin protein (SEQ ID NO. 2), molecular oxygen and the luminophore coelenterazine (Inouye et al., 1989;Johnson and Shimomura, 1978;Shimomura and Johnson, 1978). When three Ca2+ ions bind to this complex, coelenterazine is oxidized to coelenteramide, with a concomitant release of carbon dioxide and blue light (emission maximum ˜466 nm) (FIG. 7).
Because of its Ca2+-dependent luminescence, the Aequorin complex has been extensively used as an intracellular Ca2+ indicator detected by chemiluminescence assay.
Aequorin reportedly does not disrupt cell functions or embryo development (Miller et al., 1994).
Aequorin can be easily expressed in mammalian cells. It has been utilized to monitor the cytosolic-free calcium concentration (Thomas and Delaville, 1991) (Sheu et al., 1993) (Stables et al., 2000).
Aequorin can also be easily targeted to specific organelles such as mitochondria (Brini et al., 1999) (Rizzuto et al., 1992) to monitor different aspects of calcium homeostasis.
The pharmaceutical industry has taken wide advantage of the different properties of Aequorin, particularly in High Throughput Screens (Detheux, 2000). The activation of a receptor coupled to the phospholipase C transduction pathway can be easily detected in presence of the photoprotein Aequorin, because of an instantaneous release of calcium from the endoplasmic reticulum. WO0002045, Detheux et al. (EUROSCREEN S.A.) describes a high-throughput screening diagnostic and/or dosage method of an agonist and/or an antagonist for a calcium-coupled receptor (in mammalian cells) where Aequorin is used as marker for intracellular calcium changes upon receptor stimulation.
It has been previously shown that Aequorin can be functionally expressed in yeast and detected. Nakajima-Shimada et al. (Nakajima-Shimada et al., 1991b) describe the monitoring of intracellular calcium in Saccharomyces cerevisiae with an Apoaequorin cDNA expression system. Here, Aequorin was again used as a marker of intracellular calcium upon stress or glucose variations in the medium.
In contrast to mammalian signal transduction, there is no comparable Ca2+ release from the endoplasmic reticulum upon G protein-coupled receptor (GPCR) activation in yeast cells. The addition of α-factor to a yeast cell (i.e. stimulation of the GPCR Ste2) raises [Ca2+]i from a basal level of approximately 100 nM to a few hundred nanomolar in the cells, simultaneous with the induction of Ca2+ influx. When the cells are incubated with α-factor in a Ca2+-deficient medium, Ca2+ influx is greatly reduced, and the rise in [Ca2+]i is not detected (Iida et al., 1990). This slight variation in cytosolic [Ca2+] does not interfere with pathway activity detection according to the instant invention.
A limited number of reporter genes are known for use in the yeast cells, such as Saccharomyces cerevisiae, and their use is not always appropriate in screening methods. Accordingly, there is a continuing need for additional yeast reporter gene systems optimized for screening methods.
For screening purposes, a reporter gene product must be easy to detect. Accordingly, the most commonly utilized reporter gene in yeast is LacZ, which encodes the very big and stable enzyme β-Galactosidase, which is detected in a chemiluminescence assay. However, bacterial contamination may occur in yeast cultures during assays and most of the contaminants physiologically express a β-Galactosidase activity. Contaminated cultures give a very strong signal in the presence of β-Galactosidase substrates, leading to false positives in an assay.